EP2077329A1 - Cochon transgénique avec fonction incrétine altérée - Google Patents

Cochon transgénique avec fonction incrétine altérée Download PDF

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EP2077329A1
EP2077329A1 EP08000111A EP08000111A EP2077329A1 EP 2077329 A1 EP2077329 A1 EP 2077329A1 EP 08000111 A EP08000111 A EP 08000111A EP 08000111 A EP08000111 A EP 08000111A EP 2077329 A1 EP2077329 A1 EP 2077329A1
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Prior art keywords
transgenic
transgenic pig
cell
gip
diabetes mellitus
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English (en)
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Barbara Kessler
Rüdiger Wanke
Nadja Herbach
Alexander Pfeifer
Eckhard Wolf
Simone Renner
Andreas Hofmann
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Ludwig Maximilians Universitaet Muenchen LMU
MWM Biomodels GmbH
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Ludwig Maximilians Universitaet Muenchen LMU
MWM Biomodels GmbH
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Priority to EP08000111A priority Critical patent/EP2077329A1/fr
Priority to US12/348,294 priority patent/US7919673B2/en
Priority to EP09000044A priority patent/EP2077330A1/fr
Publication of EP2077329A1 publication Critical patent/EP2077329A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0276—Knock-out vertebrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/507—Pancreatic cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A01K2217/054—Animals comprising random inserted nucleic acids (transgenic) inducing loss of function
    • A01K2217/056—Animals comprising random inserted nucleic acids (transgenic) inducing loss of function due to mutation of coding region of the transgene (dominant negative)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/108—Swine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0362—Animal model for lipid/glucose metabolism, e.g. obesity, type-2 diabetes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/027—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a retrovirus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism

Definitions

  • the present invention relates to transgenic pigs containing a dominant-negative incretin hormone receptor, namely the dominant-negative human glucose-dependent insulinotropic polypeptide receptor.
  • the present invention furthermore relates to uses of these transgenic pigs as clinically relevant animal model systems for studying the development and novel therapies for diabetes mellitus type 2, particularly for the maintenance and expansion of pancreatic ⁇ -cell mass.
  • the incretin hormones glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are secreted by enteroendocrine cells in response to nutrients like fat and glucose and enhance glucose-induced release of insulin from pancreatic ⁇ -cells (1,2).
  • GIP and GLP-1 are both secreted within minutes of nutrient ingestion and facilitate the rapid disposal of ingested nutrients.
  • Both peptides share common actions on islet ⁇ -cells acting through structurally distinct yet related receptors. Incretin-receptor activation leads to glucose-dependent enhancement of insulin secretion, induction of ⁇ -cell proliferation, and enhanced resistance to ⁇ -cell apoptosis.
  • GIP also promotes energy storage via direct actions on adipose tissue, and enhances bone formation via stimulation of osteoblast proliferation and inhibition of bone resorption.
  • GIP and GLP-1 are rapidly degraded by the enzyme dipeptidyl peptidase-4 (DPP-4).
  • GIP and GLP-1 are mediated through specific 7-transmembrane-domain G-protein coupled receptors, GIPR and GLP-IR, respectively (3).
  • GIPR or GLP-1R is coupled to increases in cAMP and intracellular Ca 2+ levels, as well as activation of P1-3K, Epac 2, PKA, PKB, MAPK and phospholipase A2 and finally leads to enhanced exocytosis of insulin-containing granules (4).
  • T2D diabetes mellitus type 2
  • GIP glucose-dependent insulinotropic polypeptide
  • T2D type 2 diabetes mellitus
  • the problem is solved by the present invention by providing a transgenic pig.
  • the transgenic pig according to the present invention comprises a recombinant nucleic acid encoding a dominant-negative human glucose-dependent insulinotropic polypeptide receptor (hGIPR dn ).
  • hGIPR dn a dominant-negative human glucose-dependent insulinotropic polypeptide receptor
  • nucleic acid refers to polynucleotides, such as DNA, RNA, modified DNA, modified RNA as well as mixtures thereof.
  • a "dominant-negative" GIP receptor refers to a modified GIPR which binds the ligand GIP but is not capable of signal transduction. This dominant-negative receptor competes with the endogenous intact GIP receptor for the ligand GIP and - depending on the level of expression of the dominant-negative receptor - impairs the function of GIP.
  • the recombinant nucleic acid encodes a protein which comprises an eight amino acid deletion (residues 319-326) and an amino acid change at residue 340, preferably Ala to Glu, in the third intracellular loop of the hGIPR.
  • the recombinant nucleic acid encodes a protein comprising SEQ ID NO: 1 or having SEQ ID NO: 1.
  • the recombinant nucleic acid comprises SEQ ID NO: 2, the cDNA sequence corresponding to SEQ ID NO: 1.
  • the recombinant nucleic acid encoding hGIPR dn is comprised in a plasmid or viral vector.
  • a preferred viral vector is a lentiviral vector.
  • the expression vectors can be introduced into somatic cells which will then be used for nuclear transfer to generate cloned transgenic animals.
  • the plasmid or viral vector comprises a promoter which allows for expressing the hGIPR in the transgenic pig, preferably allows expression in the pancreatic islets, preferably the ⁇ -cells of the pancreas.
  • a preferred promoter is an insulin promoter, more preferably rat insulin 2 gene promoter (RIPII).
  • RIPII rat insulin 2 gene promoter
  • Further suitable promoters are the pig INS promoter or other promoters which confer expression in the pancreatic islets, such as PDXI.
  • promoters with other tissue specificities are used to evaluate the role of GIP receptor function in other GIP target tissues, such as adipose tissue, bone, and brain.
  • the recombinant nucleic acid is a lentiviral vector comprising the rat insulin 2 gene promoter (RIPII).
  • FIG. 1a A preferred viral construct is shown in Figure 1a .
  • the hGIPR dn is expressed in the islets of the pancreas, preferably the ⁇ -cells.
  • the hGIPR dn is overexpressed in the islets of the pancreas, preferably the ⁇ -cells, of the transgenic pig compared to wild-type hGIPR.
  • the transgenic pig according to the present invention preferably contains the recombinant nucleic acid encoding hGIPR dn is in its germ cells and somatic cells.
  • transgenic cell lines can be established by breeding to generate a standardized model system for metabolic research, particularly in the context of diabetes.
  • the recombinant nucleic acid encoding hGIPR dn is integrated into the genome of the transgenic pig.
  • the dominant-negative GIPR is expressed from an episomal vector.
  • the transgenic pig exhibits elevated postprandial glucose levels as well as distinct reduction of initial insulin secretion.
  • the transgenic pig exhibits a reduced oral glucose tolerance.
  • pancreatic islet and ⁇ -cell mass of the transgenic pig is reduced.
  • the volume densities of the islets in the pancreas (Vv (lslet/Pan) ) and of the ⁇ -cells including isolated ⁇ -cells in the pancreas (Vv ( ⁇ -cell/Pan) ) are preferably reduced in GIPR dn transgenic pigs vs. controls. Accordingly, the total islet volume (V (lslet,Pan) ) and the total volume of ⁇ -cells including isolated ⁇ -cells (V ( ⁇ -cell,Pan) ) are smaller in GIPR dn transgenic pigs compared to non-transgenic littermate controls (see also Figures 6b to d) .
  • the problem is furthermore solved by the present invention by providing a transgenic cell or transgenic cell line from a transgenic pig of the present invention.
  • transgenic cell or cell line is obtained from a transgenic pig of the invention.
  • the transgenic cell or cell line is obtained from a germ cell and/or a somatic cell of said transgenic pig.
  • transgenic cells or cell lines from the transgenic pigs.
  • transgenic cells and/or cell lines are suitable in vitro test systems and can be used for developing autologous cell replacement therapies.
  • transgenic cells and/or cell lines can be used to generate a standardized model system for metabolic research, particularly in the context of diabetes.
  • transgenic cells and/or cell lines preferably respective somatic cells/cell lines, can also be used to obtain transgenic pigs, such as by cloning strategies.
  • the problem is furthermore solved by the present invention by using a transgenic pig according to the present invention as model system for the development and therapy of diabetes mellitus, in particular diabetes mellitus type 2.
  • Diabetes mellitus type 2 (formerly called diabetes mellitus type II, non insulin-dependent diabetes (NIDDM), obesity related diabetes, or adult-onset diabetes) according to the present invention refers to a metabolic disorder that is primarily characterized by insulin resistance, relative insulin deficiency, and hyperglycemia. It is rapidly increasing in the developed world, and there is some evidence that this pattern will be followed in much of the rest of the world in coming years. Unlike type 1 diabetes, there is little tendency toward ketoacidosis in type 2 diabetes. One effect that can occur is nonketonic hyperglycemia. Complex and multifactorial metabolic changes lead to damage and function impairment of many organs, most importantly the cardiovascular system in both types. This leads to substantially increased morbidity and mortality in both type 1 and type 2 patients, but the two have quite different origins and treatments despite the similarity in complications.
  • the transgenic pigs according to the present invention are a highly suitable animal model system for diabetes mellitus because, as described herein, they exhibit the following clinical symptoms of diabetes mellitus type 2: glucose intolerance and reduction of pancreatic ⁇ -cell mass.
  • the transgenic pig model furthermore overcomes the limitations of the mouse model, as e.g. disclosed in DE 198 36 382 C2 .
  • a known transgenic mouse contains an altered GIPR which binds GIP but does not induce signalling after binding GIP.
  • the transgenic mice expressing the altered GIPR in the ⁇ -cells of the pancreas develop a severe diabetes during the first two months of their lives.
  • no specific stimulation studies with GIP or GLP-1 can be performed in the mouse model due to its limited size.
  • the unexpected severe phenotype is due to specific inhibition of the GIP effect or due to a mere unspecific disturbance of the ⁇ -cells due to a high level of overexpression of the dominant-negative receptor which may cause - in part - non-specific effects, e.g.
  • transgenic pigs of the present invention for the first time a transgenic large animal model with impaired incretin function is established.
  • the use preferably comprises the evaluation of the role of impaired GIP signalling in the pathogenesis of diabetes mellitus, in particular diabetes mellitus type 2.
  • the use preferably comprises the characterization of the mechanisms by which GIP supports pancreatic islet maintenance in vivo.
  • the use preferably comprises the development and evaluation of incretin-based therapeutic strategies of diabetes mellitus, in particular diabetes mellitus type 2.
  • diabetes mellitus type 2 in particular diabetes mellitus type 2.
  • An example is already provided by the Exendin-4 treatment study, as described herein below. Different treatment regimens can be evaluated with regard to efficacy and safety. Important readouts will be the dynamics of insulin secretion (which can only be determined with the required high resolution in time in a large animal model) and effects on pancreatic islet mass.
  • the use preferably comprises the dynamic monitoring of islet mass in diabetes mellitus patients, in particular diabetes mellitus type 2 patients.
  • said use comprises the development of novel methods for the dynamic monitoring of islet mass in patients.
  • Potential approaches combine specific in vivo labelling of pancreatic islets with state-of-the art imaging technology.
  • the problem is furthermore solved by the present invention by providing a method for identifying targets to bypass or overcome aGIPR signaling defect, comprising the utilization of a transgenic pig according to the present invention.
  • the problem is furthermore solved by the present invention by providing a method for identifying a compound that modulates the incretin hormone system or complex, comprising:
  • Preferred administration routes of a compound are oral, nasal, subcutaneous, intracutaneous, parenteral, transdermal, topical, intravenous, intraarterial, intramuscular, intraperitoneal or combinations thereof.
  • Insulin effect refers to a significantly greater insulin stimulatory effect evoked after an oral glucose load than that evoked from an intravenous glucose infusion when plasma glucose concentrations are matched.
  • the incretin effect is determined by an oral glucose tolerance test or by stimulation studies with GIP or GLP-1. For more details, see Examples.
  • promoters with other tissue specificities are used to evaluate the role of GIP receptor function in other GIP target tissues, such as adipose tissue, bone, and brain. These promoters can also be used in the methods of the invention, such as in order to identify compounds that modulate the incretin hormone system or complex in specific tissues or in a tissue-specific manner.
  • transgenic pigs expressing a dominant-negative GIP receptor (GIPR dn ) in the pancreatic islets using lentiviral transgenesis.
  • the insulinotropic effect of intravenously administrated GIP was blunted, whereas the GLP-1 receptor agonist Exendin-4 elicited a seven-fold (p ⁇ 0.05) increase in serum insulin in GIPR dn pigs.
  • GIPR dn transgenic pigs exhibited significantly elevated glucose levels (p ⁇ 0.05) as well as markedly reduced insulin secretion (p ⁇ 0.01) following oral glucose challenge, but did not develop diabetes mellitus until an age of at least 15 months.
  • pancreatic ⁇ -cell mass was reduced by more than 70% in 12-month-old GIPR dn transgenic pigs compared to littermate controls.
  • the inventors generated a large animal model mimicking the impaired GIP function in T2D patients.
  • a lentiviral vector was cloned that expresses a dominant-negative GIPR (GIPR dn ) under the control of the rat insulin 2 gene promoter (RIPII) ( Figure 1a ).
  • the GIPR dn has an eight amino acid deletion (positions 319-326) and an Ala ⁇ Glu exchange at amino acid position 340 in the third intracellular loop, which is essential for signal transduction (11).
  • Lentiviral vectors were injected into the perivitelline space of pig zygotes. A total of 113 injected zygotes were transferred laparoscopically into the oviducts of three cycle synchronized recipient gilts (sow 1: 32 zygotes; sow 2: 31 zygotes, sow 3: 50 zygotes) (12). 19 piglets (17% of the transferred zygotes) were born. Southern blot analysis identified 9 founder animals (47.3% of the born animals) carrying one or two lentiviral integrants ( Fig. 1b ), confirming the high efficiency of lentiviral transgenesis in large animals (12).
  • pancreatic islets were isolated from transgenic and non-transgenic offspring of both founder boars and analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). Expression of the GIPR dn was detected in the islets of all transgenic animals, but not in the islets of non-transgenic littermates. No signals were obtained from islets of transgenic offspring after omission of the RT step, demonstrating that expressed rather than integrated sequences were detected ( Figure 1c ).
  • RT-PCR reverse transcriptase-polymerase chain reaction
  • GIPR dn transgenic pigs exhibited a significantly reduced insulin release (52% smaller AUC insulin; p ⁇ 0.05) going along with a decelerated decline of blood glucose levels (10% larger AUC glucose; p ⁇ 0.05) ( Figure 4 ).
  • impaired GIPR function causes a general disturbance of insulin secretion and/or alterations in islet structure and/or islet integrity.
  • the total pancreas weight did not differ between GIPR dn transgenic pigs and littermate control animals.
  • the organs were divided into two portions along a clearly defined anatomical structure ( Figure 5 ).
  • the left pancreatic lobe was processed for islet isolation (14).
  • the number of islet equivalents was reduced by 93% (p ⁇ 0.05) in pancreas samples of GIPR dn transgenic pigs as compared to control pigs (Table 2).
  • Table 1 Table 1
  • Type of Pig Total pancreas weight (gram) Pancreas weight wo. left lobe (gram) wt #1 224 114 wt #2 148 80 wt #3 190 100 mean ⁇ SEM 188 ⁇ 22 98 ⁇ 10 tg #1 198 93 tg #2 190 97 tg #3 211 107 mean ⁇ SEM 200 ⁇ 6 99 ⁇ 4 Table 2 .
  • pancreatic islet profiles of GIPR dn transgenic pigs appeared to be smaller in size and reduced in number ( Figure 6a ). These findings were confirmed by quantitative stereological investigations (15) of the remnant organ.
  • the total islet volume (V (lslet,Pan) ; Figure 6b ) and the total volume of ⁇ -cells including isolated ⁇ -cells (V ( ⁇ -cell,Pan) ; Figure 6c ) were significantly (p ⁇ 0.05) smaller in GIPR dn transgenic pigs compared to non-transgenic littermate controls.
  • volume density data not shown
  • Figure 6d volume density of isolated ⁇ -cells (single insulin positive cells and small ⁇ -cell clusters containing less than five ⁇ -cell profiles, Vv (iso ⁇ -cell/Pan) , V (iso ⁇ -cell,Pan) ), a parameter indicating islet neogenesis, were not different between the two groups.
  • GIPR knockout mice (Gipr - / - ) provided no evidence that GIPR action is required for the maintenance of islet and ⁇ -cell integrity in vivo (16,17) Interestingly, these mice exhibited an increase in relative ⁇ -cell area referring to pancreas area (16), leading to the conclusion that in vivo the function of GIP is primarily restricted to that of an incretin (17).
  • the relatively mild phenotype of Gipr - / - mice may result from compensatory mechanisms (17). Islets from Gipr - / - mice were shown to exhibit increased sensitivity to exogenous GLP-1 (16).
  • double mutant mice DIRKO
  • lacking both GIPR and GLP-1R exhibited more severe glucose intolerance than the individual mutants (18,19), also these double mutant animals did not develop diabetes mellitus.
  • RIPII-GIPR dn transgenic mice develop massive early-onset diabetes mellitus with marked structural changes of the pancreatic islets (11), precluding long-term studies as to the role of GIP signaling for islet maintenance in the absence of glucose toxicity.
  • the massive changes observed in GIPR dn transgenic mice were unexpected and are possibly due to a high level of overexpression of the dominant-negative preceptor which may cause - in part - non-specific effects, e.g. by squelching of G-proteins.
  • GIPR dn transgenic pigs represent a more clinically relevant animal model for a plethora of applications in basic and translational research including the detailed characterization of mechanisms by which GIP supports islet maintenance in vivo, the development and preclinical evaluation of incretin-based therapies (reviewed in 20) as well as the development of novel methods for dynamic monitoring of islet mass in T2D patients (21).
  • the expression cassette consisting of the rat insulin 2 promoter (RIPII) and the cDNA of a dominant-negative human glucose-dependent insulinotropic polypeptide receptor (hGIPR dn ) was described previously (11) and cloned into the lentiviral vector LV-pGFP (22) via the Cla I and Sal I restriction sites. Recombinant lentivirus was produced as previously described (22).
  • the respective cDNA sequence is shown in SEQ ID NO: 2.
  • the respective amino acid sequence is shown in SEQ ID NO: 1.
  • Zygotes were collected from 6-month old superovulated and artificially inseminated gilts after slaughter as previously described (12). As recipients six-month-old estrus-synchronized gilts were used. Pig embryo transfers were performed laparoscopically as previously described (23).
  • Offspring were genotyped by PCR using DNA prepared from ear tips by application of the Wizard DNA Extraction Kit (Clontech, Mountain View, USA).
  • Transgene-specific primers were used: sense: 5'-TTT TTA TCC GCA TTC TTA CAC GG-3' [SEQ ID NO: 3] and antisense 5'-ATC TTC CTC AGC TCC TTC CAG G-3' [SEQ ID NO: 4].
  • genomic DNA (aliquots of 8 ⁇ g) extracted from EDTA blood using the Blood and Cell Culture DNA Midi kit (Qiagen, Hilden, Germany), was digested with the restriction enzyme Apa I and hybridized with a 32 P-labeled probe directed towards the RIPII promoter sequence.
  • transgene specific primers sense: 5'-TTT TTA TCC GCA TTC TTA CAC GG-3' [SEQ ID NO: 3] and antisense 5'-ATC TTC CTC AGC TCC TTC CAG G-3' [SEQ ID NO: 4].
  • Example 5 Non-surgical and surgical implantation of central venous catheters
  • the OGTT was performed in 5-month-old non-restrained, freely moving animals. After an 18-h overnight fast, animals were fed 2 g/kg BW glucose (25) mixed with 100g of commercial pig fodder (Deuka porfina U, Deuka, Düsseldorf, Germany). The meal was eaten from a bowl under supervision. Blood samples were obtained from the jugular vein catheter at -15, -5, 0, 15, 30, 45, 60, 90, 120, 150 and 180 minutes relative to the glucose load. Blood glucose was immediately determined using a Precision ® Xceed TM Glucometer (Abbott, Wiesbaden, Germany). For serum insulin measurements see below.
  • the GIP/Exendin-4 stimulation test was performed in 7-months-old (28-36 weeks) non-restrained freely moving animals. Following an 18-hour fasting period, 0.5 g glucose/kg BW were administered intravenously as a bolus of concentrated 50% glucose solution (26). After three minutes 20 pmol/kg BW of synthetic porcine GIP (Bachem, Weil am Rhein, Germany) or 10 pmol/kg BW of synthetic Exendin-4 (Bachem) were administered intravenously. Blood samples were collected -15, 0, 1, 3, 6, 9, 11, 13, 18, 23, 28, 33, 48 and 60 minutes relative to the glucose load.
  • Serum glucose levels were determined using an AU 400 autoanalyzer (Olympus, Hamburg, Germany) while serum insulin levels were measured in duplicate using a porcine insulin radioimmunoassay (RIA) kit (Millipore, Billerica, USA) according to the manufacturer's instructions.
  • RIA porcine insulin radioimmunoassay
  • Example 8 lntravenous glucose tolerance test (IVGTT)
  • the IVGTT was performed in pigs at 10 months (45 weeks ⁇ 2 weeks) of age. After an 18-h overnight fast, a bolus injection of concentrated 50% glucose solution (0.5 g glucose/kg BW) (26) was administered through the central venous catheter. Blood was collected at -15, -5, 0, 1, 3, 5, 10, 20, 30, 40, 50, 60, 120 and 180 minutes relative to the glucose load. Serum glucose and serum insulin levels were determined as described above (Example 7).
  • Example 9 Pancreas preparation and islet isolation
  • Pancreatic islets were isolated from three 12- to 13-month-old GIPR dn transgenic pigs and three littermate control animals ( ⁇ 220 kg). After precise explantation of the pancreata in toto, the major pancreatic duct was canulated using a Cavafix ® Certo 18G catheter (B. Braun). Following separation from the rest of the organ along a clearly defined anatomical structure (see Figure 5 ), the left pancreatic lobe was distended with University of Wisconsin (UW) solution containing 4 PZ units NB8 collagenase (Nordmark, Uetersen, Germany) per gram of organ and 0.7 DMC units neutral protease (Nordmark).
  • UW University of Wisconsin
  • pancreata were digested using a modification of the half-automated digestion-filtration method as previously described (27). Purification of the isolated islets was performed with the discontinuous OptiPrep TM density gradient (Progen, Heidelberg, Germany) in the COBE 2991 cell processor (COBE Inc., Colorado, USA) (28). Purified islets were cultured in HAM'S F12 culture medium (Cell Concepts, Umkirch, Germany; supplemented with 10% FCS, 1% amphotericine B, 1% L-glutamine, 1% ampicilline/gentamycine and 50 mM nicotinamide) at 24°C and 5% CO 2 in air.
  • HAM'S F12 culture medium Cell Concepts, Umkirch, Germany; supplemented with 10% FCS, 1% amphotericine B, 1% L-glutamine, 1% ampicilline/gentamycine and 50 mM nicotinamide
  • Islet numbers were determined using dithizone (DTZ) stained islet samples 29 (triplicates) which were counted under an Axiovert 25 microscope (Zeiss, Oberkochen, Germany) with a calibrated grid in the eyepiece, grouped into size categories and converted into islet equivalents (IEQ), i.e., islets with an average diameter of 150 ⁇ m. Purity of the islets was estimated by two independent individuals using dithizone stained samples of the islet suspension (triplicates).
  • DTZ dithizone stained islet samples 29 (triplicates) which were counted under an Axiovert 25 microscope (Zeiss, Oberkochen, Germany) with a calibrated grid in the eyepiece, grouped into size categories and converted into islet equivalents (IEQ), i.e., islets with an average diameter of 150 ⁇ m. Purity of the islets was estimated by two independent individuals using dithizone stained samples of the islet suspension (triplicates).
  • islet vitality For determination of islet vitality, an aliquot of the islet suspension was stained with freshly prepared fluorescein diacetate (FDA, Sigma) and propidium iodide solution (PI, Sigma) and then estimated by two independent individuals using a BX50 fluorescence microscope (Olympus).
  • FDA fluorescein diacetate
  • PI propidium iodide solution
  • pancreas was cut into 1 cm thick slices. Slices were tilted to their left side and covered by a 1 cm 2 point-counting grid.
  • tissue blocks were selected by systematic random sampling, fixed in 10% neutral buffered formalin, routinely processed and embedded in paraffin. From a series of approximately 4 ⁇ m thick sections, one section was stained with hematoxylin and eosin (H&E) and the following section was used for immunohistochemistry.
  • the volume of the pancreas (V (Pan) ) before embedding was calculated by the quotient of the pancreas weight and the specific weight of the pig pancreas (1.07 g/cm 3 ).
  • the specific weight was determined by the submersion method (30).
  • the indirect immunoperoxidase technique was applied to localize insulin containing cells as previously described (11).
  • a polyclonal guinea pig anti-porcine insulin antibody (dilution 1:1,000) (Dako Cytomation, Hamburg, Germany) as well as horseradish peroxidase conjugated polyclonal rabbit anti-guinea pig IgG (dilution 1:50 containing 5% (vol/vol) porcine serum) were used.

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CN110305872A (zh) * 2019-07-17 2019-10-08 中国农业科学院北京畜牧兽医研究所 小型猪2型糖尿病模型的构建方法及应用
US20210100225A1 (en) * 2015-10-08 2021-04-08 President And Fellows Of Harvard College Multiplexed Genome Editing
US12058986B2 (en) 2017-04-20 2024-08-13 Egenesis, Inc. Method for generating a genetically modified pig with inactivated porcine endogenous retrovirus (PERV) elements

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US10126220B2 (en) * 2013-07-22 2018-11-13 National Oilwell Varco, L.P. Systems and methods for determining specific gravity and minerological properties of a particle
ES2784643T3 (es) 2014-08-28 2020-09-29 Univ Aarhus Modelo de cerdo para la diabetes
CN113699116A (zh) 2014-12-10 2021-11-26 明尼苏达大学董事会 用于治疗疾病的遗传修饰的细胞、组织和器官
CN106222176A (zh) * 2016-08-08 2016-12-14 中国农业科学院深圳农业基因组研究所 一种dna分子及其在制备糖尿病鼠模型中的应用

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210100225A1 (en) * 2015-10-08 2021-04-08 President And Fellows Of Harvard College Multiplexed Genome Editing
US12058986B2 (en) 2017-04-20 2024-08-13 Egenesis, Inc. Method for generating a genetically modified pig with inactivated porcine endogenous retrovirus (PERV) elements
CN110305872A (zh) * 2019-07-17 2019-10-08 中国农业科学院北京畜牧兽医研究所 小型猪2型糖尿病模型的构建方法及应用

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